Horizontal gene transfer in Clostridium difficile strains
About Clostridium difficile is a significant nosocomial pathogen and the etiological agent of antibiotic-associated diarrhea. This bacterium produces two potent toxins, known as toxin A and B. These primary virulence factors are chromosomally encoded on a region called the pathogenicity locus (PaLoc), which also contains regulatory genes. This region of DNA is absent in Clostridium difficile strains that are non-toxigenic. It has been shown that the PaLoc can be transferred from toxigenic strains to three non-toxigenic strains of different ribotypes. A transconjugate is shown by a cytotoxicity assay to produce toxin B at a similar level to that of the donor strain. This in return demonstrates how a toxogenic strain can pass virulence through horizontal gene transfer. Materials and methods All strains that were used in this study were grown in brain-heart infusion media supplemented with 5% defibrinated horse blood. These were grown under anaerobic conditions (C02) at 37 °C. Maintaining these growth conditions are important because it allows the bacteria to thrive, as well as mimic the environment consistent with that of a human host. Once a sufficient incubation period was obtained, the bacterial DNA was isolated using a Gentra Puregene Yeast/Bact. Kit, and the DNA was then amplified using PCR. The cells were then washed, and pellets were taken up into BHI broth as donor and recipient cultures were mixed. Cellular mixtures were transferred into 0.45 micro liter pore sized cellulose nitrate filters. Once incubated for 24 hours together, the culture was vortexed and spread into selective plates. This was done in an effort to determine donor, recipient and transconjugate cell numbers on the filters. The presence of the PaLoc gene region within the chromosomal DNA of the cells was determined using another PCR reaction with the tcdA-F primers. The presence of this gene locus enabled researchers to determine the presence of either the A or B toxin in previously non-toxigenic strains. PaLoc transfer to non-toxigenic C. difficile CD37 To make the determination of weather genetic elements were cotransferred in the absence of direct selection, nine CD37 transconjugates containing an an erythromycin-resistant derivative were initially investigated by PCR and subsequent DNA sequencing. One of the strains had unexpectedly acquired the PaLoc. To determine weather the PaLoc could be transferred independently, a genetically marked PaLoc was used as a donor with CD37 as a recipient. PaLoc containing transcojugates were obtained at a frequency of 7.5 × 10−9 transconjugants per donor. It comes important however to also determine if the newly acquired toxin genes are capable of directing toxin production. These genes may be present, yet the genomic evolution in the recipients is less significant if these genes are non-functional. To do this, a PaLoc containing transconjugate was designated PaLoc386. This strain was subject to a cytotoxicity assay with HFF-1 cells (from human fibroblast cell line). The recipant cells were incubated from 24 hours with a monolayer of HFF-1 cells before the cytopathic effect was determined. After incubation the cellular supernatants were collected, and it was determined that PaLoc386 produced similar levels of toxin to the donor strain. This shows that when in contact with human cells acting as a stimulant for the bacterium, the recipients do in fact posses a functional PaLoc derived from horizontal gene transfer. HGT on variable sized DNA fragments The mechanism underlying the process of the PaLoc gene transfer was determined using whole-genome sequences. This was done for seven of the transconjugates containing either the marked or wild type PaLoc. To accurately define the horizontally transferred regions in the transconjugates, researchers needed to distinguish between donor and recipient genomes. Thirty regions of about 10kb even spaced about the donor genome were selected and compared with corresponding regions of the CD37 genome. This region was selected on the basis that they did not contain any mobile genetic elements (ie transposons, prophages etc.). The presence of 10-150 SNPs allowed for discrimination between donor and recipient DNA. Through Alignment of the genomic DNA sequences of the PaLoc regions, researches found regions 1-3 and 8-30 to be derived from the recipient. In all transconjugates, the PaLoc is located between regions 6 and 7. It was also found that the PaLoc has transferred on variable size DNA fragments, either between regions 6 and 7 from the donor strain in 6 out of 7 transconjugates, and regions 5 and 6 from the donor in transconjugant PaLoc385. In the transconjugate PaLoc26, the PaLoc was transferred on a relatively small DNA fragment (max size of 67,644bp). This contrasts with other cotransfers, which occurred on separate DNA fragments. The length of the fragments of transferred DNA were determined by calculating the distance of the first SNP upstream of the PaLoc to last SNP downstream of the PaLoc that was specific to the donor strain. Due to the fact that there was a high level of sequence identity between CD37 and the donor in the regions surrounding the PaLoc, this study could only determine that maximum and minimum size of the transferred fragment. Altogether, the PaLoc was transferred on fragments of DNA ranging from at least 63,034bp to a max of 272,977 bp. This may affect the versatility and ease of transfer of genes to non-toxigenic strains. Donor DNA fragments Resources 1. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826655/#!po=25.0000 Brouwer, Michael S.M., Adam P. Roberts, Haitham Hussain, Rachel J. Williams, Elaine Allan, and Peter Mullany. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 17 Oct. 2013. Web. 15 Nov. 2014.] 2. [http://www.atcc.org/products/all/SCRC-1041.aspx "HFF-1 (ATCC® SCRC-1041™)." HFF-1 ATCC ® SCRC-1041™ Homo Sapiens The Cell Line Was Estab. N.p., n.d. Web. 15 Nov. 2014.]